Plant Tissue Culture technique | PTC definition, steps, scope and application

Plant Tissue Culture (PTC), a sophisticated biotechnological tool, has revolutionized the propagation, conservation, and genetic improvement of plants. PTC involves the in vitro cultivation of plant cells, tissues, or organs in a controlled environment, providing researchers with the ability to manipulate and study plant growth and development at the cellular level

Definition

Plant tissue culture broadly refers to the in-vitro cultivation of plants, seeds, plant parts (tissues, organs, embryos, single cell , protoplast etc.) on solid or liquid nutrients media under aseptic conditions. 

General Terminology used 

• Explant –  parts of the plant or cell used for the culture 
• Callus – It is the un-organized mass of plant cells (Parenchymatous nature). And the process of generating these types of mass of cells is known as Callogenesis.
• Organogenesis – the production of plant organs (roots or shoots) from explant.
• Micropropagation – development and multiplication of whole plant from explant in laboratory.

History of plant tissue culture

In 1902, Gottlieb Haberlandt, an Austrian botanist, attempted first to culture isolated somatic cells  (green, photosynthetic cells from leaf bract mesophyll of Lamium purpureum) of higher plants in vitro with the intention to study the properties and potentialities of the cell. He is also known as the father of Plant Tissue Culture. 

In 1922, knudson – Asymbiotic germinated orchid seed in vitro and Robbins culture root tip in vitro.

In 1934, White successfully cultured the root tip of tomato for unlimited time in medium (liquid) containing inorganic salts, 2% sucrose, and 0.01% yeast extract.

In 1939, Gautheret, Nobecourt  and white successfully established a continuous growing callus culture (discovery of callus generation).

In 1941, Van Overbeek demonstrated the first time in Datura that coconut milk contains factors essential for growth and development.

In 1952,  Morel & Martin developed meristem culture to obtain virus free Dahlias.

In 1953, Tulecke produced the haploid callus of the gymnosperm Ginkgo biloba from pollen

In 1954, Muir et al – first plant from single cell

In 1955, Miller et al – Discovered kinetin as a cell division hormone

In 1957, Skoog and miller discovered that organ formation can be regulated by changing the ratio of auxins and cytokine.

In 1962, Murashige and Skoog developed murashige & skoog nutrition medium for PTC.

In 1964, Production of First haploid embryos from pollen grain of Datura innoxia by Guha & Maheshwari

In 1971, Takabe et al., Regenerated of first plants from the protoplast of tobacco.

In 1972, Carlson et al – first report of interspecific hybridization through protoplast fusion in two species of tobacco.

Steps of Plant Tissue Culture Process:

1. Pre-propagation – Selection of appropriate plant , Pretreatment of plant- It is the first step in the plant tissue culture. To select the plant-  characteristics traits such as disease resistance, high yield, or specific biochemical properties, Plant Age (preferablly Young and actively growing tissues), Cultural History (pesticide or herbicide treatments) and purity (free from pathogens, contaminants, and diseases) should be checked to avoid introducing issues into the tissue culture process.
2. Preparation of nutrient medium – Nutrient medium is mixture either liquid or semi-solid of important substance e.g. macroelements,  micro  elements,  amino acids,   vitamins,   iron   source,   carbon   source like sucrose   and   phyto-hormones.
   • Different media compositions are used for different tissues or plants or based on the objective. 
   • Example of different PTC medium – MS-medium, B5 medium, White medium etc.
3. Selection of explants (shoot tip, meristem tip, nodal bud, floral meristem and bud) – Once we have selected the plant and medium of choice for PTC, depending on the objective, specific parts of the plant are then separated by cutting for culture. For example, for the development of virus-free plants, shoot/meristem tips are used. This separated portion of the plant, which we would use, is called an explant.
4. Surface sterilization – This explant is further treated with various disinfectants/ surfactants (e.g. sodium hypochlorite, ethanol, sterile distilled water, Tween 20 etc.) to remove microbial contaminants on the surface of explants in this step. 
5. Inoculation of explant – Surface sterilized explants are transferred into the flask or jar containing culture medium in this step.
6. Propagation or Development  of  plants  in the growth  room:- plants need specific light and dark periods, temperature etc.for their proper development. For example, the generation of 16 hours of light and 8 hours of darkness  with Temperature (20-25 0C) is generally required.
   • Subculturing- transfer of cultured cell/tissue/plant into fresh medium. This step is also necessary for Prevention of Senescence and Deterioration, Adjustment of Growth Conditions, Elimination of Contaminants, Control of Overgrowth etc.of culture.
7. Hardening  of  micro  plants (Ex vitro adaptation or plant acclimatization)– In this step plants are transferred into small pots (in green houses )and eventually into the field. 

Scope of Plant tissue culture

Plant tissue culture can be used in a range of fields of study. For example,-

• Propagation: Plant tissue culture is extensively used for the mass production of plants, allowing rapid and efficient propagation of selected and elite plant varieties.
• Germplasm Conservation: Tissue culture provides a method for the conservation and preservation of plant genetic material. Rare or endangered plant species can be maintained in vitro to prevent their extinction.
• Genetic Modification: Tissue culture techniques play a crucial role in genetic engineering and the development of genetically modified plants with desirable traits such as resistance to pests, diseases, or tolerance to environmental stresses.
• Research and Study: It serves as a valuable tool for studying plant growth, development, and physiology. Researchers can manipulate environmental conditions more precisely in a controlled in vitro environment
• Production biomolecule/metabolite: Certain plants/ plant tissues can be induced to produce secondary metabolites of pharmaceutical or industrial interest in controlled environments.

Certification of tissue culture raised plants

Though by micropropagation of tissue culture of plants a large number of plants can be produced in a short time, there are risks of, Pathogens that  are often symptom less,  such  as viruses, pose a risk of spread of disease and variations that affect yield through somaclonal variations  associated with the micropropagated cultured plant. This could lead to the loss of productivity of crop plant or plant cultured.

This  concern requires a well structured system to maintain standard tissue culture to ensure virus free quality planting material for commercial production. 

In India for the above issue, the Department   of   Biotechnology   (DBT), Government of India has  established the National Certification System for Tissue Culture Raised Plants (NCS  TCP).  

Application of plant tissue culture

Plant tissue culture can be applied purposes like – 

• Micropropagation: Mass production of plants through the culture of small plant parts, such as shoot tips or nodal segments, to generate numerous identical plants.
• Somatic Embryogenesis: Induction of embryos from somatic cells, leading to the development of somatic embryos that can be used for plant regeneration.
• Callus Culture: Culturing undifferentiated cells (callus) that can be used for plant regeneration or for the production of cells for genetic transformation.
• Embryo Culture: In vitro culture of embryos at various stages of development for purposes such as rescue of hybrid embryos or in vitro fertilization.
• Anther and Pollen Culture: Culture of anthers or isolated pollen grains to induce androgenesis, leading to the development of haploid plants.
• Protoplast Isolation and Culture: Isolation of plant cells devoid of cell walls (protoplasts), followed by their regeneration into whole plants.
• Virus Elimination: Treatment of infected plant materials to obtain virus-free plants.
• Transgenic Plant Production: Introduction of foreign genes into plant cells, followed by the regeneration of transgenic plants expressing the desired traits. E.g.
  • BT-Cotton – The Cotton plant carry gene of Bacillus thuringiensis (BT gene), which gives plant insecticidal properties.
  • Flavr-Savr tomato (Transgenic Tomato) – polygalacturonase (PG gene) carrying tomato. This gene helps to improve the quality and shelf life of tomatoes.

Reference

Sussex I. M. (2008). The scientific roots of modern plant biotechnology. The Plant cell, 20(5), 1189–1198. https://doi.org/10.1105/tpc.108.058735

García-Gonzáles, R.,Quiroz K.,  Carrasco B.,  Caligari P. Plant tissue culture: Current status, opportunities and challenges. Cien. Inv. Agr. 37(3):5-30. 2010

Espinosa-Leal, C. A., Puente-Garza, C. A., & García-Lara, S. (2018). In vitro plant tissue culture: means for production of biological active compounds. Planta, 248, 1-18.

Bhoite, H. A., & Palshikar, G. S. (2014). Plant tissue culture: A review. World journal of pharmaceutical sciences, 565-572.

Ikeuchi, M., Sugimoto, K., & Iwase, A. (2013). Plant callus: mechanisms of induction and repression. The Plant cell, 25(9), 3159–3173. https://doi.org/10.1105/tpc.113.116053